By induction and analysis of mutants of cultured rodent cells, we hope to contribute to our understanding on the genetics and biochemistry of regulation of pyrimidine metabolism in mammals, including man. Using a previously isolated pyrimidine auxotrophic mutant of Chinese hamster cells, as well as additional auxotrophs, revertants and cell hybrids, we plan to investigate genetic complementation, dominance relationship, and the number and chromosomal locations of the structural genes for enzymes of the pyrimidine biosynthetic pathway. Biochemical and immunological characterization of mutant and wild type enzymes may reveal the organization and coordinate regulation of the two multienzyme complexes of this pathway. The possible existence of multiple control mechanisms, such as repression, sequential induction, and feedback inhibition of enzyme activities will be studied by use of genetic and/or chemical blocks of the pathway. A statistical approach to gene mapping by analysis of gene assortment in cell hybrids may enable us to identify and locate such regulatory genes responding to the changing levels of intracellular pyrimidine metabolites. In addition, this and other types of regulatory mutants of the pyrimidine pathway will be isolated by two methods: the first involves screening for cells overproducing intermediary metabolites and thus able to crossfeed a pyrimidine-requiring indicator strain, and the second involves direct selection for those resistant to enzyme inhibitors. Finally, a combined mutational and biochemical approach will be taken to elucidate the catabolism and interconversion of pyrimidines.